This invention relates to sphingosine kinase, which catalyzes phosphorylation of sphingosine to form sphingosine-1-phosphate (SPP) and to molecules encoding sphingosine kinase, including mutants, variants, fragments and derivatives thereof, to vectors and transfected host cells which express sphingosine kinase. The present invention also relates to methods for evaluating stimulatory or inhibitory effects of agents on the sphingosine kinase production and activity.
Sphingosine-1-phosphate (SPP), a sphingolipid metabolite, which regulates diverse biological processes, such as cell growth, differentiation, survival, motility, and calcium mobilization, is now emerging as a new member of a class of lipid signaling molecules with dual intra and intercellular actions. This phosphorylated derivative of sphingosine, the structural backbone of all sphingolipids, has many of the hallmarks of classical second messengers. The level of SPP is very low in cells and is rapidly increased by activation of sphingosine kinase (SPHK), the enzyme responsible for the formation of SPP. SPHK is a member of a highly conserved gene family and is distinct from other known lipid kinases. Changes in SPHK activity is induced by diverse physiological stimuli, including platelet-derived growth factor (PDGF), nerve growth factor (NGF), muscarinic acetylcholine agonists; TNF-a, activation of protein kinase C (PKC), and cross-linking of the immunoglobulin receptors. Like to other signaling molecules, SPP has a short half life due to rapid turnover catalyzed by SPP lyase and/or SPP phosphatase. Inhibition of SPP formation by competitive inhibitors of SPHK blunted the mitogenic response to PDGF, the cytoprotective effects of NGF, vitamin D3, PKC, and cAMP activators. Furthermore, calcium mobilization induced by FceR1, FcgR1, and muscarinic acetylcholine receptors are affected by SPP formation.
Because sphingosine-1-phosphate (SPP), acts both intracellularly and extracellularly to affect many biological processes, including mitogenesis, apoptosis, atherosclerosis and inflammatory responses, it is necessary to develop means of stimulating or inhibiting formation of SPP. Specific members of the EDG-1 family of G protein-coupled receptors bind SPP and modulate chemotaxis, angiogenesis neurite reaction and cell rounding. Because SPP levels are mainly regulated by the activity of SPHK, cloning and characterization of this enzyme are important for identifying normal and pathological processes. Hence, availability of the product of the invention as a means of evaluating effect on SPHK and SPP levels and activity is important for use in identifying agents that would inhibit or stimulate SPHK activity.
The SPHK has been purified from rat kidneys and subsequently identified in mouse cDNAs encoding two forms of SPHK, designated mSPHK1a and mSPHK1b, whose predicted proteins differ by only 10 amino acids at their N-terminus. Furthermore, the human SPHK cDNA (hSPHK) has now been defined. The corresponding mRNAs may arise by alternative splicing.
It is the purpose of this invention to provide SPHK, including human SPHK, and antibodies thereto for use in research, for use in diagnosis and for use in identifying agents which will inhibit or enhance activity of SPHK. The SPHK (using comparative control samples) and antibodies thereto may be used in diagnostic kits measuring the level of SPHK. SPHK activity is implicated as a contributing factor in several disease conditions, including cancer, stroke, atherosclerosis, inflammatory responses, allergic responses (including asthma) and Alzheimer""s. The discovery of this invention makes it possible to test effect of drug candidates on inhibition and stimulation of activity of SPHK. Cell-free SPHK in a biologically non-toxic carrier can be added to substrates to evaluate drug candidates"" effect on activity of hSPHK. It would, therefore, be possible to evaluate and propose dosages for drugs might either inhibit or stimulate SPHK activity.